NF-.kappa.B is a transcription factor that plays a pivotal role in the highly specific pattern of gene expression observed for immune, inflammatory and acute phase response genes, including interleukin 1, interleukin 8, tumor necrosis factor and certain cell adhesion molecules. Like other members of the Rel family of transcriptional activators, NF-.kappa.B is sequestered in an inactive form in the cytoplasm of most cell types. A variety of extracellular stimuli including mitogens, cytokines, antigens, stress inducing agents, UV light and viral proteins initiate a signal transduction pathway that ultimately leads to NF-.kappa.B release and activation. Thus, inhibitors and activators of the signal transduction pathway may be used to alter the level of active NF-.kappa.B, and have potential utility in the treatment of diseases associated with NF-.kappa.B activation.
An important modulator of NF-.kappa.B activation is the inhibitor protein I.kappa.B.alpha., which associates with (and thereby inactivates) NF-.kappa.B in vivo. Stimulus-induced phosphorylation of I.kappa.B.alpha. at serine residues (S32 and S36) renders the inhibitor a target for ubiquitination and subsequent degradation by the proteosome, leading to NF-.kappa.B activation. The induction of I.kappa.B.alpha. phosphorylation is a critical step in NF-.kappa.B activation, and the identification of I.kappa.B.alpha. kinase, as well as proteins that modulate its kinase activity, would further the understanding of the activation process, as well as the development of therapeutic methods.
Several protein kinases have been found to phosphorylate I.kappa.B.alpha. in vitro, including protein kinase A (Ghosh and Baltimore, Nature 344:678-682, 1990), protein kinase C (Ghosh and Baltimore, Nature 344:678-682, 1990) and double stranded RNA-dependent protein kinase (Kumar et al., Proc. Natl. Acad Sci. USA 91:6288-6292, 1994). Constitutive phosphorylation of I.kappa.B.alpha. by casein kinase II has also been observed (see Barroga et al., Proc. Natl. Acad. Sci USA 92:7637-7641, 1995). None of these kinases, however appear to be responsible for in vivo activation of NF-.kappa.B. For example, phosphorylation of I.kappa.B.alpha. in vitro by protein kinase A and protein kinase C prevent its association with NF-.kappa.B, and phosphorylation by double-stranded RNA-dependent protein kinase results in dissociation of NF-.kappa.B. Neither of these conform to the effect of phosphorylation in vivo, where I.kappa.B.alpha. phosphorylation at S32 and S36 does not result in dissociation from NF-.kappa.B.
Other previously unknown proteins with I.kappa.B.alpha. kinase activity have been reported, but these proteins also do not appear to be significant activators in vivo. A putative I.kappa.B.alpha. kinase was identified by Kuno et al., J Biol. Chem. 270:27914-27919, 1995, but that kinase appears to phosphorylate residues in the C-terminal region of I.kappa.B.alpha., rather than the S32 and S36 residues known to be important for in vivo regulation. Diaz-Meco et al., EMBO J 13:2842-2848, 1994 also identified a 50 kD I.kappa.B kinase, with uncharacterized phosphorylation sites. Finally, Chen et al, Cell 84:853-862, 1996 identified a kinase that phosphorylates I.kappa.B.alpha., but that kinase was identified using a non-physiological inducer of I.kappa.B.alpha. kinase activity and requires the addition of exogenous factors for in vitro phosphorylation.
Accordingly, there is a need in the art for an I.kappa.B.alpha. kinase that possesses the substrate specificity and other properties of the in vivo kinase. There is also a need for improved methods for modulating the activity of proteins involved in activation of NF-.kappa.B, and for treating diseases associated with NF-.kappa.B activation. The present invention fulfills these needs and further provides other related advantages.